Voltage-gated sodium channels regulate the rapid and specific flow of sodium ions through the cell membrane. They are of great importance for such functions in the human body as the regulation of the heartbeat and electrical signaling in nerve cells. Examples of diseases caused by mutations in sodium channels include fatal cardiac arrhythmias, epilepsy, neuromuscular disorders and severe migraines. Furthermore, sodium channels are also promising targets in the treatment of pain and potentially in the prevention of cancer metastasis. Sodium channels are the target for a vast array of natural toxins, many of them highly selective animal peptide toxins. These toxins represent a treasure trove of bioactive compounds with potential applications as tools for basic sodium channel research as well as in the development of drugs for the treatment of sodium channel- related diseases. Spider toxins that target sodium channels do this indirectly through binding to the voltage sensor domains (VSDs) and shifting the channel activation voltage. These toxins are of special interest because of their potential to control channel activity in a subtle and controlled way with high specificity. Current knowledge of the mode of action of gating-modifier toxins is mostly based on functional and mutational studies with very little direct structural information available. Obtaining structural information has been hampered by the large size of eukaryotic sodium channels (> 2000 amino acid residues) and the general difficulties in working with membrane proteins. In this project, an isolated bacterial sodium channel VSD (from the halophile Bacillus halodurans) reconstituted into phopholipid bicelles will be used as a model system for investigating channel-toxin interactions at the atomic level using solution nuclear magnetic resonance (NMR) spectroscopy. A protocol for the production of isotopically labeled VSD samples suitable for NMR structural studies has been established, and most of the necessary NMR spectra for making essentially complete backbone assignments have been collected. A VSD-binding toxin from chilean rose tarantula (Grammostola rosea) has been identified (VSTx2) and will be used in the interaction studies. The full-length B. halodurans sodium channel has also been expressed in HEK cells, and patch-clamp functional studies of this channel have been performed. During the proposed project period, solution-state NMR will be used to identify the interacting residues on VSD and toxin and to characterize the conformational changes that the toxin induces upon binding. With this information, a model for the toxin-channel complex and the mechanism of action of the toxin will be developed. This hypothesis will then be functionally verified by site-directed mutagenesis followed by patch-clamp electrophysiology of the full channel. Lastly, the foundation for future studies into toxin specificity will be laid by using the recombinant bacterial VSD or bacterial-human chimeric VSD proteins to identify additional gating- modifier toxins from the venom of different tarantula species. All of this information will assist the development of drugs targeting ion channels to treat disorders ranging from epilepsy to genetic heart disease to pain.